This paper introduces a highly sensitive microwave biosensor based on a defected ground structure (DGS) for label-free detection of minute volume liquid samples. The biosensor incorporates a resonant structure etched into the ground plane, which concentrates the electric field to form a strongly localized sensing “hot-spot”. This configuration significantly enhances sensitivity to minute variations in the dielectric properties of tiny volume samples. Using MEMS fabrication techniques, the DGS biosensor is integrated with a microfluidic channel capable of single-cell trapping, thereby enabling high-precision measurements at the single-cell level. An equivalent circuit model is developed and simulated to quantitatively correlate the sample’s dielectric constant with the resonant frequency. Experimental results demonstrate that the biosensor achieved frequency shifts on the order of MHz in tests involving microplastic particles and various tumor cell types, highlighting its high detection sensitivity and discrimination capability. Furthermore, a dual-branch DGS configuration is designed to enable dual-band sensing, and simulations are conducted to analyze the influence of microsphere dielectric properties on both resonant frequencies. The fabricated biosensor exhibits consistent performance across particles and cell-based assays, offering a viable and effective platform for the highly sensitive detection of minute volume biological samples.